Place
samples in metal sample holder (boat) which has immersed in acetone (using mesh
basket to separate
samples when necessary; covered with mesh stripe-mat), taking care to prevent the tissue samples from drying.
Load the specimen boat and then screw the door back on.

Samples
should be pre-fixed in acetone.

Samples
can be arranged as desired in various metal baskets available in drawer labeled
“CPD”.

Turn on
cold water.

Water
should fill the jacket around the drying chamber.

This
cools the chamber to 15-20oC. Hence,
as CO2 is let into the chamber it is a liquid, rather than a gas.

Water
should come out of the reddish rubber hose in the sink.

Open the black knob (#)1 to let CO2
into the chamber.

Make
sure all 3 black knobs on the CPD are closed and the valve on the CO2 tank
is open.

Then
open the black knob on top of the CPD nearer the window of the CPD, knob 1.

There is liquid CO2in the chamber visible in the
window on
the back of the chamber. Look through the window (using the mirror on
the wall if you want). The liquid CO2
can be faint and difficult to see.

Open the black knob (#3) to let CO2
out of the chamber using the black knob nearer under the chamber, knob 3.

The CO2 level
should stay high enough to cover the sample. This prevents damage to the sample.

Turn
off the cold water or leave it trickle..

Flush the apparatus as outlined above (step 4 to 5)
every half- or 1-hour for 30 sec or so, depending on the size of specimen, to allow specimens to infiltrate with
CO2 and remove dehydrating fluid. Large specimens will require about 2-3 hr total time, while small
specimens will be done in about 1 hr. Remember to leave the room door
wide open to dissipate the CO2 gas that is being
vented.

After flushing, close the inlet valve and lower the liquid level to just below the top of the boat by venting off excess gas.

Turn on the hot water (while leaving the
cold water partially on if necessary).

Wait while the temperature and pressure rise.

This
can take a few mins.

The
temperature starts to rise ahead of the pressure.

Once the critical point (31.5oC and 1200 psi (lb/in2)) has been exceeded, shut off the hot tap water, and wait for 1-5 min depending on the size of specimen. Wait
longer (up to 0.5-1 hour) for larger and/or thicker samples (hot tap water is required sometimes).

Open the knob 2 on the top of the chamber to
allow the gaseous CO2 to escape slowly.

Be sure
to let the CO2 out gradually.

It
should take 10-20 min for the pressure to reach zero.

Open the door (sometimes it takes a while for the door to be opened), remove the sample and store
the samples into sealed glass vials.

Be gentle with
the sample as dry material can be brittle.

Make sure the CO2 valve on cylinder is closed after finish.

Background (http://www.polaron-range.com/)

Critical Point Drying is an
established method of dehydrating
biological tissue prior to examination in the Scanning Electron Microscope
(SEM).

The phase diagram shows the pressure to temperature ranges where solid, liquid
and vapor exist. The boundaries between the phases meet at a point on the phase
diagram called the triple point.
Along the boundary between the liquid and vapor phases it is possible to choose
a particular temperature and corresponding pressure, where liquid and vapor can
co-exist and hence have the same density. This is the critical temperature and
pressure, or critical point.

Critical point drying relies on
this physical principle by bring samples to the critical of a suitable inert
fluid.This inert fluid replaces water
in the sample, and then vaporizes at its critical point without change of
density and therefore without surface tension effects which distort morphology
and ultrastructure. CO2 is universally used today as the inert
fluid.

The critical point of CO2
is at approximately 31.5oC
and 1200 psi (lb/in2) or 78 bar.

Since liquid CO2 is not
sufficiently miscible with water, it is necessary to use an intermediate fluid (such as acetone, which is miscible with both water and liquid CO2) during this process.